The long-term goal of our research is to develop critically-needed small molecule drugs to help treat the approximately 33 million people worldwide living with HIV. The emergence of deleterious drug-resistance mutations against currently-approved therapies necessitates new strategies for targeting HIV life-cycle events that include complementary inhibition mechanisms and exploitation of regions with high sequence conservation. An innovative approach, recently developed in our lab, aims to leverage the wealth of energetic and structural information inherent to atomic-level molecular footprints - interaction maps occurring within targetable pockets on viral proteins - to rationally identify, develop, and design novel small molecule inhibitors against the viral protein gp41. As outlined below, we have strong evidence suggesting that footprints derived from regions on gp41 encode information regarding which residues are most critical for ligand binding and that our development of a novel computational means to harness this information is a potentially ground-breaking way to screen for, or alternatively, design-from-scratch small molecule fusion inhibitors. Here, the objectives are to utilize the power of footprints to rationally design small molecules that specifically bind to gp41 and inhibit membrane fusion. Our published atomic models for peptide inhibitors (C34 and T20/Fuzeon) with gp41 will enable us to target the known hydrophobic pocket in addition to other regions that, until now, have yet to be exploited. Our central hypothesis is that small organic molecules which make interactions with gp41, in an energetically similar manner as key side-chains on known peptide inhibitors, will make effective leads for therapeutics. Furthermore, by including footprints derived from interactions occurring within conserved gp41 regions during the identification and development stages, we postulate compounds will be more likely to have robust resistance profiles. This hypothesis is based on several observations, including our identification of seven experimentally-verified inhibitors usin a new virtual screening protocol tailored to account for footprint-based similarity. We have also used footprints in conjunction with molecular dynamics simulations to explain the origins of T20 drug resistance as well as show that association of C-helix peptide inhibitors can be driven solely by changes within the conserved gp41 pocket, supporting the premise that the conserved pocket region is an important drug target site. Promising preliminary results, in which we have integrated footprints with computational de novo design methods, facilitates, for the first time, custom construction from scratch of small molecules which make specific footprint patterns with gp41 in a similar way as a known reference. In Aim #1 we will custom-design small molecule inhibitors specifically tailored to energetically favorable interfaces on gp41. In Aim #2 we will develop experimentally verified gp41 leads to have improved gp41 activity. In Aim #3 we will identify other targetable events in the gp41 pre-hairpin model.